thanks for developing an amazing tool like DESeq2 and having it available to the scientific community. I am new to RNAseq analysis. I have read the vignettes and I have some questions about my dataset. I looked online but I could not find an answer similar to my question.
I have a study of two animal genotypes, control and genetically altered and these animals received treatment or left untreated. So given these conditions I have control treated, control untreated, genetically altered treated and genetically altered untreated. I have collected tissue samples from different areas of the gut as I am looking on colorectal cancer (small intestine and large intestine).
My questions are as follows: 1) Shall I have two datasets for my analysis (i.e. one for large intestine and one for small intestine)? I did the RNAseq run of all the samples. I tried to have everything in DESeqDataSetFromMatrix but the design gets very complicated. I am interested only in each tissue separately (i.e. either small intestine or large intestine) and not in their differences. 2)My second question has to do with the design. I have noted gender, parent, batch which can all be confounding variables. Shall I include in the design or use sva to remove hidden batch effects? If I use sva, shall I have the confouding factors in my initial design formula and then change according to sva (I can use as reference the code given in the vignette)? I am not sure if gender and parent have an effect.
I am providing an example of the design I have used and it worked. For more complicated designs, I get an error for “Model matrix not full rank”
#dds_colon<-DESeqDataSetFromMatrix(countData=countdata_colon,colData=coldata_colon, design= ~Gender + Genotype +Treatment+ Genotype:Treatment)
3)My third and last question has to do with one part of my analysis. For some samples I got tumours and healthy tissue, so they are paired samples. However not all the samples are paired. Shall I create a separate column in metadata for this comparison or shall I create a different model?
I would like to thank you in advance for all your suggestions and advice. I know some of my questions sound a bit basic but I am completely lost on how to proceed.